Project Summary The goal of this Phase I SBIR is to develop a new platform that increases the stability and activity lifetime of oxidase enzymes used in amperometric biosensor fabrication. This platform is based upon Design-Zyme?s new, patent-pending approach for in vitro glycosylation of proteins and enzymes. This Phase I proposal consists of two specific aims designed to (1) demonstrate that Design-Zyme?s novel platform can be leveraged to enhance and improve the stability and activity lifetime of many, if not all, oxidase enzymes, and (2) to immediately commercialize three improved enzymes for use by university, governmental, and industrial entities. Our approach will be extended in Phase II to other protein families potentially useful in sensing applications, e.g. dehydrogenase enzymes. The field of biosensors has developed into a practical, cost-effective, and portable screening modality with proven animal and human applications in health monitoring, disease diagnosis, drug development, food safety, and point-of-care diagnostics. Our enhanced enzymes will enable the development of biosensors that have been heretofore unavailable, which is expected to positively impact the development and manufacturing of devices for new sensing applications. These new devices, based on the enzyme system(s) developed here, will also provide a new paradigm for the clinical monitoring of glucose tolerance, blood-lactate monitoring, and other disease states relevant to integrative medicine and ultimately, human health. These enhanced enzymes, and the underlying platform, are a necessary first step to the development of continuous in vivo analyte monitoring lasting months or years with little outside intervention. This proposal is based on our significant preliminary results showing that in vitro glycosylation does not compromise enzyme activity, but instead can enhance an enzyme?s initial activity and lifetime. This is in contrast to most existing methods of glycosylation. We have found that hyaluronic acid, when covalently attached to an oxidase, can improve its activity lifetime in solution and provide extended protection at elevated temperatures. The stabilized enzymes that are a product of this Phase I can find utility in any monitoring device that presently uses oxidase enzymes. This includes amperometric biosensors, wearable patches and other measurement modalities. At the end of the Phase II SBIR, we will have a fully optimized platform that can optimize any oxidase enzyme for use in in vivo (human and animal) biosensors.